应用表达载体介导siRNA抑制胰腺癌skp2基因表达的研究

Studies on the Inhibition of siRNA Expression Vector Mediated skp2 Expression in Human Pancreatic Cancer Cells

目的探讨表达载体介导小干扰RNA(siRNA)对人胰腺癌细胞系Capan-2skp2基因表达的抑制作用。方法构建靶向SKP2的siRNA质粒表达载体,采用LipofectamineTM2000转染Capan-2细胞,实时定量PCR和Western blot观察Capan-2细胞转染后SKP2mRNA和蛋白表达的改变。结果PCR和DNA测序证实SKP2siRNA表达载体构建成功,分别转染Capan-2细胞后,skp2基因的mRNA和蛋白表达量与对照组相比均明显下降(P<0.05)。结论所构建的靶向SKP2的siRNA质粒表达载体可以有效地阻断Capan-2细胞SKP2的mRNA和蛋白表达,为下一步以SKP2为靶点的胰腺癌的基因治疗实验研究奠定基础。

Objective To construct a series of eukaryotic expression vector which can transcript shRNA against human skp2 gene and investigate the inhibitory effect of siRNA on skp2 expression in pancreatic caner cell line Capan-2. Methods Three pairs of hairpinlike oligonucleotides specific for human skp2 gene were designed and synthesized. The annealed oligonucleotides designed contain 5＇- BamH I and 3＇-EcoR I overhangs. Then they could be ligated into ＂the eukaryotic expression RNAi-Ready pSIREN-RetroQ vector. The recombined plasmids were verified by digestion with restriction endonuclease. Results The shRNA sequences were successfully inserted into the eukaryotic expression vector pSIREN-RetroQ. The sequence-specific siRNA efficiently down regulated the SKP2 expression at both mRNA and protein levels. The inhibition rate was 64.16% at mRNA level and 60.26% at protein level, respectively. Conclusion Three siRNA expression vectors were successfully constructed. The siRNA plasmid expression vectors against SKP2 can inhibit the SKP2 expression in Capan-2 cells efficiently. RNAi-mediated gene silencing of SKP2 can be a novel modality in gene therapy of pancreatic cancer.